1.

Introduction

Gold nanoparticles (Au NPs), typically with sizes of 0.3 to 110 nm, are widely studied due to their remarkable optical, electrical, and chemical properties.^1–4 Conventional Au NPs, bigger than 2 nm, exhibit an extremely high density resulting in collective oscillation of free electrons under excitation with light, also known as surface plasmon resonance.^5–7 The ultrasmall size of photoluminescent gold nanoclusters (Au NCs) comparable to the Fermi wavelength of electrons ($\sim 0.7\mathrm{nm}$) induces distinctive quantum confinement effects,^8–11 which result in unique molecule-like behaviors due to discrete energy levels. Over the past decade, photoluminescent Au NPs, composed of several atoms to a few nanometers, have been developed and have stimulated extensive interest.¹² Low toxicity and facile synthesis under eco-friendly conditions make photoluminescent Au NCs a very promising alternative to organic fluorophores and semiconductor quantum dots (QDs) in broad ranges of biomedical research and applications. Under certain conditions, UV light is used to initiate synthesis of Au NPs.^13–16 On the other hand, UV photons have high energies and might induce other effects on already synthesized Au NPs, inducing their decomposition. There are very few reports on the photoluminescence stability of Au NCs under irradiation with UV light.^17–20 Some authors have reported photoluminescence instability of dimethylformamide (DMF) protected Au NCs;¹⁷ however, prolonged studies showed that the photostability of DMF-protected Au NCs is higher compared to trypsin stabilized Au NCs.¹⁸ There are also papers showing that the photostability of photoluminescent Au NCs coated with dihydrolipoic acid (DHLA) overcomes the photostability of organic fluorophores, such as fluorescein and rhodamin 6G, but yields to the photostability of hydrophilic polymer-coated CdSe/ZnS QDs.¹⁹ One of the most photostable was ribonuclease-A-encapsulated Au NCs²⁰ which leads to the conclusion that the photostability of photoluminescent Au NCs varies according to the coating material. Despite the inert nature of gold and the reported stability of Au NPs, using Au NPs for bioimaging might cause their degradation under continuous excitation with UV/blue light,²¹ resulting in toxicity. It should be stressed that photostability is the major characteristic of any marker for microscopy, optical imaging, and biopsy. Therefore, the study of photostability and photoluminescence bleaching under continuous excitation with UV/blue light has to be investigated before applying photoluminescent Au NCs for the imaging of biological objects.

In this paper, we present a comprehensive study on the spectroscopic characteristics and photostability of photoluminescent 2-($N$-morpholino) ethanesulfonic acid (MES)-coated Au NCs under irradiation with near-ultraviolet and visible light.

2.

Materials and Methods

2.2.

Synthesis of Au-MES NPs

The Au NPs capped with MES were synthesized according to the modified synthesis protocol of Bao et al.:²² 5 ml of aqueous MES buffer solution (1 M, pH 6.3, the pH value was achieved using NaOH) was mixed with 1 ml of chloroauric acid solution (0.29 M). Synthesis was performed under vigorous stirring for 21.5 h at the temperature of 37°C.

After the synthesis solution, the Au NPs were centrifuged for 30 min (10,000 rpm or $g=6.7\times {10}^3$) with a “MiniSpin plus” centrifuge (WTW GmbH, Germany).

The synthesized colloidal solution of MES-coated Au NPs exhibited a deep red color in the daylight and greenish blue photoluminescence under UV light (Fig. 1 inset).

Fig. 1

Absorption, photoluminescence (${\lambda }_{\mathrm{ex}}=420\mathrm{nm}$), and PL excitation (${\lambda }_{\mathrm{em}}=476\mathrm{nm}$) spectra of Au-MES NPs; absorption spectra of chlorauric acid and MES buffer solution. Arrows represent the wavelengths of irradiation used in photostability measurements. Inset shows images of Au-MES NPs solution in the daylight and under illumination with UV light.

3.

Results

3.2.

Photostability of Au-MES NPs

Under irradiation at around the maximum of the photoluminescence excitation band of Au-MES NCs (${\lambda }_{\mathrm{irr}}=402\mathrm{nm}$), a slight decrease of absorbance of Au-MES NPs in the spectral region from 250 to 550 nm was detected [Fig. 2(a)] as the accumulated irradiation dose increased. Absorption difference spectra revealed several distinct bleaching absorption bands with maxima at 290, 330, 366, 420, and 470 nm. Bleaching absorption bands at 290, 330, 366, and 470 nm corresponded to the bands in the absorption spectrum of Au-MES NPs. However, no correlation in intensity of the absorption bands and the bleaching bands under irradiation with a 402 nm wavelength light was observed. In addition, the bleaching absorption band at 420 nm was indistinguishable in the absorption spectrum and correlated with the main photoluminescence excitation band of Au-MES NCs. Simultaneously, decrease of intensity of PL band (${\lambda }_{\mathrm{ex}}=405\mathrm{nm}$) with a maximum at 476 nm and hypsochromic PL band shift by 47 nm which resulted from the formation of a new PL band with a maximum at 430 nm was observed [Fig. 2(b)]. No PL band shift and only a decrease in the PL intensity at 476 nm was observed when the 420 nm excitation wavelength was used [Fig. 2(c)].

Fig. 2

Spectral changes of Au-MES NPs at ${\lambda }_{\mathrm{irr}}=402\mathrm{nm}$ as accumulated irradiation dose increases. (a) Changes of absorption spectrum (top) and absorption difference spectra (bottom). PL excitation spectrum (${\lambda }_{\mathrm{em}}=476\mathrm{nm}$) (dashed curve). (b) Changes of photoluminescence spectrum (${\lambda }_{\mathrm{ex}}=405\mathrm{nm}$). (c) Photobleaching of photoluminescence spectrum (${\lambda }_{\mathrm{ex}}=420\mathrm{nm}$). Arrows represent the changes in absorbance and PL intensity.

Under irradiation of Au-MES NPs colloidal solution with a 366 nm wavelength light, the most intensive decrease in absorbance was observed at around 330 and 470 nm wavelengths (Fig. 3). Distinct photobleaching bands with maxima at 366 and 420 nm wavelengths were observed in absorption difference spectra. Only the bleaching absorption bands at around 330 and 470 nm corresponded to the absorption bands of Au-MES NPs. PL changes were similar to the ones observed under irradiation with the 402 nm wavelength light (data not shown).

Fig. 3

Changes of absorption spectrum (top) and absorption difference spectra (bottom) of Au-MES NPs at ${\lambda }_{\mathrm{irr}}=366\mathrm{nm}$ as accumulated irradiation dose increases. Arrow represents the changes in absorbance.

By irradiating Au-MES NPs solution with a near-UV light at 330 nm, the most intensive decrease of the absorbance was detected at around 300 and 470 nm (Fig. 4). At the same time, an increase of the PL (${\lambda }_{\mathrm{ex}}=405\mathrm{nm}$) intensity was observed (Fig. 4 inset), although the peak position remained intact. The bleaching band at 300 nm in the absorption difference spectra did not coincide with the one at 330 nm which was observed under irradiation at 366 nm (Fig. 3) indicating that each irradiation wavelength (${\lambda }_{\mathrm{irr}}=366$; 330 nm) affected a different fraction of the nonluminescent gold NPs.

Fig. 4

Changes of absorption spectrum (top) and absorption difference spectra (bottom) of Au-MES NPs at ${\lambda }_{\mathrm{irr}}=330\mathrm{nm}$ as accumulated irradiation dose increases. PL excitation spectrum (${\lambda }_{\mathrm{em}}=476\mathrm{nm}$) (dashed curve). Inset—changes of PL spectrum (${\lambda }_{\mathrm{ex}}=405\mathrm{nm}$). Arrows represent the changes in absorbance and PL intensity.

When irradiating a colloidal solution of Au-MES NPs with a 470 nm wavelength light, no changes in absorption and PL spectra were observed even when the maximum irradiation dose of $\sim 100\mathrm{J}/{\mathrm{cm}}^2$ was accumulated (data not shown).

4.

Discussion

Metal NPs of a size on the same order as the electron Fermi wavelength ($\sim 0.5\mathrm{nm}$ for silver and gold)^23–25 often exhibit strong single electron excitations and emit photoluminescence.¹ Synthesized according to the modified synthesis protocol of Bao et al.²² colloidal MES-coated Au NPs exhibited a deep red color in the daylight and greenish blue photoluminescence under near-UV excitation (Fig. 1 inset). The absorption spectrum of the synthesized colloidal solution did not coincide with the PL excitation spectrum (${\lambda }_{\mathrm{em}}=476\mathrm{nm}$) indicating that there are not only photoluminescent Au NCs but also some nonluminescent substance in the synthesized solution. The PL excitation spectrum had a main maximum at 420 nm and another band of lower intensity at around 260 nm. Absorption bands at 290, 330, 366, and 470 nm correspond to absorption of the nonluminescent Au-MES NPs. Nevertheless, there is no absorption in the spectral region above 510 nm that would indicate the existence of Au NPs exhibiting localized surface plasmon resonance (LSPR).^5–7 According to the literature, the NPs that are bigger than 4 nm in diameter exhibit LSPR.^5,6 In contradiction to that, up to 10 nm and up to 6 nm diameter Au NPs were detected using atomic force microscopy and TEM, respectively (Fig. 5). The discrepancy between those results could be due to differences of the two techniques, as TEM gives us only the size of the core of the Au-MES NPs and atomic force microscopy gives the size of the NPs, which is several nanometers bigger than the core itself due to the MES coating. From our measurements, we can clearly state that several distinct types of Au-MES NPs could be distinguished: very small ($<1\mathrm{nm}$) photoluminescent Au NCs (observed only with AFM), and other Au NPs below 10 nm in diameter.

Fig. 5

(a) Topography of Au-MES NPs on a mica surface (AFM image) and (b) STEM image of Au-MES NPs on a carbon film.

Four different wavelengths were selected for irradiation of Au-MES NPs. Irradiation at 402 nm was preferentially absorbed by photoluminescent Au-MES NCs as the irradiation wavelength is in the range of their PL excitation band. Absorption by other nonluminescent gold NPs at 402 nm was minimal due to the minimum absorbance of the sample at this wavelength (Fig. 1). The appearance of the bleaching band at 420 nm in the absorption difference spectrum [Fig. 2(a)] that coincides with the maximum of the PL excitation spectrum and the simultaneous decrease of PL intensity at 476 nm [Figs. 2(b) and 2(c)] revealed that photoluminescent Au-MES NCs were irradiation-disrupted. However, a hypsochromic shift of the PL band position and the appearance of the new PL band at 430 nm suggested that under irradiation with 402 nm wavelength light a new type of photoluminescent NCs with a PL peak at the 430 nm wavelength were formed [Fig. 2(b)].

Following a free electron gas (Jellium) model,^23,26 a number of gold atoms $N$ in small Au NCs can be calculated from the PL band position with a simple equation

Irradiating Au-MES NCs with a 402 nm wavelength light, the discrete hypsochromic shift of the PL band revealed the formation of a photoproduct—smaller photoluminescent Au NCs of particular size. However, so far there are no other results published claiming that light would reduce the size of photoluminescent Au NCs, though a decrease of the PL intensity has been reported for DMF-, trypsin-, and DHLA-stabilized Au NCs under irradiation with near-UV light.^17–19 In contrast, irradiation of photoluminescent semiconductor QDs with UV/Vis light is well known to affect PL quantum yield^27–29 and induce a continuous hypsochromic^27,28 or bathochromic²⁹ shift of the PL band indicating photoetching and aggregation of QDs, respectively. However, a discrete spectral shift of Au-MES NCs suggesting a molecule-like behavior³⁰ complicates comparison of photoluminescent Au NCs with semiconductor QDs that undergo a continuous shift of the PL band under irradiation with UV/Vis light.^27–29

Absorption difference spectra revealed that under irradiation with 402 nm wavelength light, absorbance decreased not only at the 420 nm wavelength, but a slight photobleaching of the absorption was detected in all spectral range from 250 to 550 nm [Fig. 2(a)] showing that bigger nonluminescent NPs were also affected by irradiation. When irradiating the sample solution at ${\lambda }_{\mathrm{irr}}=330\mathrm{nm}$ at the main absorption peak not related to photoluminescent Au NCs, a decrease of absorbance at around 300 nm and 470 nm wavelengths was observed (Fig. 4) indicating the decomposition of nonluminescent Au-MES NPs. However, the shape of absorption difference spectrum did not coincide with the shape of the absorption spectrum of Au-MES NPs (Fig. 6) showing that under irradiation at 330 nm, only part of the nonluminescent Au-MES NPs were disrupted. The most sensitive to the irradiation at 330 nm were Au-MES NPs with a strong absorption at 300 nm (I type). The new bleaching absorption band with a maximum at 330 nm appeared, irradiating the sample solution at a wavelength slightly shifted to the red spectral range (${\lambda }_{\mathrm{irr}}=366\mathrm{nm}$) (Fig. 3). This absorption band could be assigned to another type (II type) of the nonluminescent Au-MES NPs which have a main absorption band at 330 nm wavelength. Discrepant changes in absorption difference spectra under irradiation at different wavelengths suggests that the absorption spectrum of Au-MES NPs is a superposition of the absorption of photoluminescent Au-MES NCs and of the minimum two different fractions of nonluminescent Au-MES NPs.

Fig. 6

Inverted normalized absorption spectrum of Au-MES NPs (black) and normalized absorption difference spectra of Au-MES NPs under irradiation at 330 nm (dark gray) and at 366 nm (light gray).

An increase in PL intensity at 476 nm that occurred simultaneously with the decrease of absorbance under irradiation at 330 nm (Fig. 4) must have occurred due to the disruption of bigger nonluminescent Au-MES NPs and formation of photoluminescent NCs from their remains as the PL intensity in the PL excitation spectrum at 330 nm is negligible and irradiation did not directly affect photoluminescent Au-MES NCs.

The low-power irradiation (20 mW) used in our experiments could not induce high energy related processes such as ablation of solid materials producing NCs that can be achieved using high-energy pulsed-lasers.^31–34 However, it has been shown that low-power irradiation with UV light can induce formation of Au NPs from chlorauric acid.¹⁴ Irradiation of Au NPs (17 to 23 nm in diameter) with low-power UV light can also induce generation of one or a few reactive oxygen species (ROS) such as hydroxyl radicals (•OH), singlet oxygen (${}^1{\mathrm{O}}_2$), superoxide radicals (${\mathrm{O}}_2^{•-}$).^35,36 Despite the fact that Zhang et al. have shown that under UV exposure of Au NPs (17 to 23 nm in size) no gold ions were detected,³⁶ upon irradiation with 402 nm (as with 366 nm) wavelength light, the size of the photoluminescent Au NCs ($<1\mathrm{nm}$ in diameter) decreased, and irradiation at 330 nm disrupted the nonluminescent NPs ($<10\mathrm{nm}$ in diameter). The greater effects on Au-MES NPs such as disruption and a decrease in size could have appeared due to increased ROS generation since under irradiation with x-rays, smaller Au NPs generate more ROS than bigger NPs due to increased surface area to volume ratio.³⁵

5.

Conclusions

In summary, the synthesized photoluminescent Au-MES NCs are not photostable under irradiation with UV/blue light. Upon irradiation at 402 nm, the photoluminescent Au-MES NCs ($<1\mathrm{nm}$ in diameter) (${\lambda }_{\mathrm{em}}=476\mathrm{nm}$) transform into a new type of photoluminescent NCs with an emission band maximum at ${\lambda }_{\mathrm{em}}=430\mathrm{nm}$. The calculations show that the size of the photoluminescent NCs decreases from $\sim 9$ to $\sim 7$ gold atoms. Moreover, nonluminescent gold NPs are also nonphotostable: disrupting the I type of nonluminescent gold NPs under irradiation at 330 nm, the photoluminescent Au NCs with an emission band maximum at ${\lambda }_{\mathrm{em}}=476\mathrm{nm}$ were formed. However, irradiation at 366 nm to the absorption of the II type of nonluminescent Au-MES NPs resulted only in the disruption of photoluminescent and nonluminescent gold NPs. The spectral changes of Au-MES NPs upon irradiation with UV/blue light could be related to the generation of ROS. Further research is required to fully investigate the photoinduced processes in NPs under low-power irradiation.